Chlamydia species are obligate intracellular parasites that are important human pathogens. C. trachomatis is the leading cause of sexually transmitted diseases and a key cause of preventable infertility in the western world, and the major cause of non-congenital blindness in developing nations. C. pneumoniae is an important cause of human respiratory infections and has been associated with the development of atherosclerotic disease. Chlamydia species have a unique intracellular life cycle but understanding its details and the mechanisms of disease pathogenesis has been hampered by the difficulty in growing the organism and the lack of genetics. In this R21 exploratory grant, we propose a novel forward genetic screen to identify host factors required for successful infection. RNA interference mediated gene inactivation provides a new approach to easily inhibit the expression of most genes, resulting in functional knockouts. It offers a novel approach to carry out large-scale forward genetic screens. Drosophila melanogaster, with its relatively small and non-redundant genome, provides an ideal "genetic" host in which to study these processes. Drosophila readily takes up small interfering RNA, allowing efficient inactivation of genes in whole flies as well as in Drosophila tissue culture cells. Several groups, including at UCSF, have had spectacular success carrying out genome-wide RNAi-mediated forward genetic screens in Drosophila tissue culture cells. Drosophila larvae and tissue culture cells have recently been used as a model system to understand the pathogenesis of several important human pathogens. In preliminary experiments, we have established that C. trachomatis infection of Drosophila tissue culture cells mimics key aspects of initial Chlamydia-mammalian cell interactions. Our long term goal is to understand how Chlamydia causes disease in humans. Our short-term goals are to identify host genes required for Chlamydial pathogenesis. Specific Aim 1: We will use RNA-mediated gene inactivation in Drosophila S2 cells to systematically inactivate all phylogentically conserved genes to identify host genes required for C. trachomatis infection. We will define the detailed phenotype and dissect the mechanism of action of each gene required for successful binding, entry, and intracellular development of C. trachomatis. Specific Aim 2: We will validate the screen by inactivating the human homologs in HeLa cells and will further investigate the role of these host genes in C. trachomatis infection. Together, these studies will significantly advance our knowledge of infectious disease pathogenesis and pave the way for new therapies and vaccines.